Xanthan Gum Capped Zno Microstars As a Promising Dietary Zinc Supplementation

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Article Xanthan Gum Capped ZnO Microstars as a Promising Dietary Zinc Supplementation

Alireza Ebrahiminezhad 1,2, Fatemeh Moeeni 2, Seyedeh-Masoumeh Taghizadeh 2, Mostafa Seifan 3, Christine Bautista 3, Donya Novin 3, Younes Ghasemi 2,* and Aydin Berenjian 3,*

1 Department of Medical Nanotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz 71348, Iran; [email protected] 2 Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz 71348, Iran; [email protected] (F.M.); [email protected] (S.-M.T.) 3 School of Engineering, Faculty of Sciences and Engineering, University of Waikato, Hamilton 3216, New Zealand; [email protected] (M.S.); [email protected] (C.B.); [email protected] (D.N.) * Correspondence: [email protected] (Y.G.); [email protected] (A.B.)

Received: 7 February 2019; Accepted: 26 February 2019; Published: 2 March 2019

Abstract: Zinc is one of the essential trace elements, and plays an important role in human health. Severe zinc deficiency can negatively affect organs such as the epidermal, immune, central nervous, gastrointestinal, skeletal, and reproductive systems. In this study, we offered a novel biocompatible xanthan gum capped zinc oxide (ZnO) microstar as a potential dietary zinc supplementation for food fortification. Xanthan gum (XG) is a commercially important extracellular polysaccharide that is widely used in diverse fields such as the food, cosmetic, and pharmaceutical industries, due to its nontoxic and biocompatible properties. In this work, for the first time, we reported a green procedure for the synthesis of ZnO microstars using XG, as the stabilizing agent, without using any synthetic or toxic reagent. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM) were used to study the structure, morphology, and size of the synthesized ZnO structures. The results showed that the synthesized structures were both hexagonal phase and starlike, with an average particle size of 358 nm. The effect of different dosages of XG-capped ZnO nanoparticles (1–9 mM) against Gram-negative (Escherichia coli) and Gram-positive (Bacillus licheniformis, Bacillus subtilis, and Bacillus sphaericus) bacteria were also investigated. Based on the results, the fabricated XG-capped ZnO microstars showed a high level of biocompatibility with no antimicrobial effect against the tested microorganisms. The data suggested the potential of newly produced ZnO microstructures for a range of applications in dietary supplementation and food fortification.

Keywords: xanthan gum; ZnO microparticles; ZnO microstars; bacteria; supplementation; food fortification

1. Introduction Zinc plays many essential roles in all life forms [1], and is part of the major subgroup of micronutrients that are beneficial in human nutrition and health [2]. In the human diet, the main sources of zinc are animal products [3], rice, wheat, and soybean [4]. However, there has been an insufficient dietary supply of the element, which has become the primary cause of zinc deficiency. Studies have revealed that the concentrations of zinc mineral elements in edible parts have decreased over the last 50 years [5], and that high levels of dietary inhibitors may suppress the absorption of

Foods 2019, 8, 88; doi:10.3390/foods8030088 www.mdpi.com/journal/foods Foods 2019, 8, 88 2 of 10 zinc [6]. Zinc deficiency has an effect on organ systems such as the epidermal, gastrointestinal, central nervous, immune, skeletal, and reproductive systems [7]. The literature has also determined that zinc deficiency can impair physical growth, and zinc-dependent metabolic functions [2]. It is essential for an individual to consume a certain level of zinc, as the zinc deficiency can have several consequences on human health. There are currently four main approaches to increasing the intake of zinc in the human diet: (1) direct supplementation, (2) food fortification, (3) dietary diversification, and (4) crop biofortification [5]. To compensate for insufficient zinc intake, the major suggested route is through the intake of oral zinc supplementation. The forms of zinc that are frequently used in supplements are zinc sulfate, zinc oxide, zinc acetate, and zinc gluconate [8]. However, zinc supplements are not well tolerated by all patients, even in high doses, due to inadequate intestinal zinc absorption [6]. Currently, there is great attention on the use of micro and nanoparticles to fortify micronutrients, due to their ultra-small size which aids intestinal absorption. Micronization is another effective technique to increase the digestive absorption of poorly soluble nutrients and drugs. It has been reported that the decrease in the particle size, up to a few microns, can substantially enhance the rate of dissolution and can consequently improve the bioavailability and clinical efficacy [9]. Although, in contrast to metal salts, which are commonly prescribed, metal and metal oxide particles are more tolerable for patients, and therefore, higher dosages can be used [6]. Nanometer-sized particles of zinc oxide (ZnO) have many applications in various areas due to their unique and superior chemical and physical properties compared to bulk ZnO. Over the last decade, massive investigations have also been performed to evaluate the effect of ZnO nanoparticles on the growth of microbial communities. For example, Ramesh et al. [10] investigated the antibacterial activity of green-synthesized ZnO nanoparticles with an average diameter of 29.79 nm. The zone inhibition method was used to explore the antibacterial activity of ZnO nanoparticles against Gram-positive (Staphyloccus aureus) strains, and Gram-negative (Salmonella paratyphi, Vibrio cholerae, Escherichia coli) strains. Authors have concluded that the ZnO nanoparticles exhibit good antibacterial activities by decreasing the cell growth. Banoee et al. [11] conducted a similar study which explored the antibacterial activity of ZnO nanoparticles with an average size of 20–45 nm against S. aureus and E. coli. It was observed that the presence of fabricated ZnO nanoparticles enhanced the antibacterial activity of ciprofloxacin, and increased the inhibition zone by 27% and 22%, for S. aureus and E. coli, respectively. However, the availability of 500 µg/disk ZnO nanoparticles decreased the antibacterial activity of amoxicillin, penicillin G, and nitrofurantoin in S. aureus. The antibacterial behavior of Vitex negundo extract assisted ZnO nanoparticles against S. aureus (ATCC 11632) and E. coli (ATCC 10536), and was studied by Ambika et al. [12] using the agar well diffusion method. The results showed that synthesized ZnO nanoparticles have high antibacterial activity. It was shown that the availability of ZnO nanoparticles contributed to a zone of inhibition of 19 mm and 16 mm for S. aureus and E. coli, respectively. To be considered as a supplement or to be used for food fortification, the fabricated ZnO must have no negative effect on gastrointestinal microbiota. Intestinal bacteria play a key role in both digestion and regulation of the immune system. For example, they are responsible for maintaining immune and metabolic homeostasis, and protecting against pathogens [13]. There are different chemical, nutritional, and immunological factors that can affect the population of human microbiota. In this regard, the properties of metallic elements such as ZnO can significantly limit gastrointestinal bacterial growth. Moreover, it is obvious that antimicrobial properties of the metallic particles can be varied by changing their physicochemical properties. In this context, the shape, particle size, and capping material are the most effective parameters [14,15]. To date, there are no reports on the successful fabrication of biocompatible ZnO particles for application as a dietary supplement. Therefore, this study was performed to synthesize a biocompatible ZnO particle using a natural heteropolysaccharide, xanthan gum (XG), as a stabilizer, thickener, and emulsifier without using any toxic chemicals. In contrast to the currently available zinc supplements, the fabrication of biocompatible micro-sized ZnO particles may be a promising Foods 2019, 8, 88 3 of 10 form of zinc supplementation, as the reduction of metal elements to a few hundreds of microns, Foods 2019, 8, x FOR PEER REVIEW 3 of 10 can substantially enhance their intestinal absorption.

2. Materials and Methods

2.1. Chemicals The zinc zinc acetate acetate dehydrate dehydrate was was obtained obtained from from Merck Merck Millipore Millipore (Darmstadt, (Darmstadt, Germany). Germany). The TheAmmonium Ammonium hydroxide, hydroxide, XG and XG andLB broths LB broths (Lysogeny (Lysogeny broth) broth) were were purchased purchased from from Sigma-Aldrich Sigma-Aldrich (St. (St.Louis, Louis, Missouri, Missouri, USA). USA). All the All solutions the solutions used usedfor the for fabrication the fabrication of nanoparticles of nanoparticles were prepared were prepared using ultrapureusing ultrapure water waterthrough through a Millipore a Millipore water water purifi puriﬁcationcation system system (Milli-Q, (Milli-Q, Milford, Milford, MA, MA, USA). USA). Ampicillin discs (10 µµg)g) and blankblank paper discs (6 mm diameter) were obtained from Fort Richard Laboratories (Auckland, New Zealand).

2.2. General Procedure for the SynthesisSynthesis of XG-Capped ZnO Microstars

2+2+ The concentrations concentrations of of Zn Zn, XG,, XG, and and NH NH4OH,4OH, along along with with the reaction the reaction time timeand temperature, and temperature, were werechosen chosen based based on preliminary on preliminary studies studies as they as theyaffected affected the size the sizeand andshape shape of the of theresulting resulting particles. particles. In 2+ 2+ theIn the first first step, step, Zn(OAc) Zn(OAc)2·2H2O·2H (as2O Zn (as source, Zn source, 1 g) and 1 XG g)and (0.2 XGg) were (0.2 dissolved g) were dissolved in 140 mL in ultrapure 140 mL waterultrapure at room water temperature. at room temperature. Then 1.5 mL Then ammonium 1.5 mL ammonium hydroxide hydroxide (NH4OH, (NH25%)4 OH,was added 25%) was dropwise added todropwise the solution. to the solution.As schematically As schematically shown in shown Figure in 1, Figure the mixture1, the mixture was refluxed was refluxed for 6 forh at 6 80 h at°C, 80 and◦C, cooledand cooled to stop to the stop reaction. the reaction. After Afterthat, the that, produc the productt was centrifuged was centrifuged and washed and washed with deionized with deionized water. water.Then, 90 Then, mL 90 of mL ultrapure of ultrapure water water was wasadded added and and the themixture mixture was was refluxed refluxed for for 9 9h. h. The The resulting material was washed with deionized water and dried in an ovenoven atat 6060 ◦°CC for 24 h.

Figure 1. Schematic illustration of the steps involved in the synthesis of xanthan gum (XG)-capped zinc oxide (ZnO) microstars using an efﬁcientefficient hydrothermalhydrothermal method.method.

2.3. Material Characterization Different analytical techniques were used to visualize and characterize the synthesized ZnO microstars. In In order to analyze the size and morphologymorphology of the fabricatedfabricated microstars, transmission electron microscopy microscopy (TEM) (TEM) (Philip (Philips,s, Eindhoven, Eindhoven, The The Netherlands, Netherlands, CM10, CM10, HT HT100 100KV) KV)was wasused. used. The Theverification veriﬁcation of the of XG-capped the XG-capped ZnO particles ZnO particles was pe wasrformed performed using Fourier using Fourier transform transform infrared infrared (FTIR) (FTIR)spectroscopy spectroscopy (PerkinElmer (PerkinElmer Spectrum Spectrum One, Waltha One,m, Waltham, Massachusetts, Massachusetts, USA). The USA). analysis The analysis was carried was −1 outcarried using out KBr using pellets, KBr and pellets, the anddata thewere data collected were collectedfor a range for of a range4000–400 of 4000–400cm−1. X-ray cm diffraction. X-ray (XRD)diffraction analysis (XRD) was analysis performed was performedto characterize to characterize the synthesized the synthesized microstars microstars using a Siemens using aD5000 Siemens X- rayD5000 powder X-ray diffractometer. powder diffractometer. The data acquisitio The datan acquisition was carried was out carriedfor an exploration out for an explorationrange (2θ degree) range ◦ (2ofθ 10–80°.degree) of 10–80 .

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2.4. Microorganism and Bacterial Culture Conditions The following bacteria were revived according to the procedure provided by the New Zealand culture collection: Escherichia coli (NZRM 250), Bacillus licheniformis (ATCC 9789), Bacillus subtilis (ATCC 9799) and Bacillus sphaericus (ATCC 4525). All freeze-dried bacteria were revived in Luria-Bertani medium by incubating the culture overnight at 37 ◦C and 200 rpm.

2.5. Determination of Antimicrobial Activity of XG-Capped ZnO Microstars Prepared ZnO microstars were suspended in distilled water and constantly treated with an ultra sonicator (Qsonica-Q800R, Qsonica, Newtown, Connecticut, USA), in an ultrasonic amplitude of 75% (500 W), until a uniform colloidal suspension was formed, which yielded a 9 mM stock solution. Serial dilution was then carried out, which resulted in 1, 3, 6, and 9 mM of freshly prepared XG-capped ZnO particle suspensions. The antimicrobial activities of the suspensions were investigated by studying the inhibition zones (agar disk-diffusion) on a comparative basis, against both Gram-negative and Gram-positive bacteria [16]. Brieﬂy, 400 µL of each grown bacteria was spread over the entire agar plate surface. Then, the blank ﬁlter paper disks were soaked in each ZnO suspension and placed in the center of the plate. A blank disc, and an ampicillin disc (10 µg), with a diameter of 6 mm, was used as a negative and positive control, respectively. The plates were incubated at 37 ◦C for 24 h. The plates were examined for evidence of zones of inhibition and the diameter was measured using a millimeter ruler.

3. Results and Discussion

3.1. Synthesis of ZnO Microstars The zinc oxide nanoparticle is an inorganic compound widely used in various applications, and can be synthesized through different methods, such as mechanochemical, controlled precipitation, precipitation in the presence of surfactants, emulsion, microemulsion, sol-gel, microwave assisted, solvothermal, and hydrothermal processes [17]. In this study, we used the hydrothermal method to fabricate XG-capped ZnO microstars, as it offered a straightforward and environmentally friendly technique without additional processing of the product such as grinding or calcination. The particle growth process can be expressed according to the following reactions:

− 2+ Zn(CH3COO)2·2H2O → 2CH3COO + Zn + 2H2O (1)

− − CH3COO + H2O → CH3COOH + OH (2) + − NH3 + H2O ↔ NH4 + OH (3) 2+ + 2+ Zn + 4NH4 → [Zn(NH3)4] (4) 2+ − Zn + 2OH → Zn(OH)2 (5) 2+ − 2− Zn + 4OH → Zn(OH)4 (6) − 2− Zn(OH)2 + 2OH → Zn(OH)4 (7) 2− − Zn(OH)4 → ZnO + 2OH + H2O (8) 2+ − [Zn(NH3)4] + 2OH → ZnO + 4NH3 + H2O (9) In the ﬁrst step, the zinc acetate reacted with ultrapure water and the corresponding zinc and acetate ions were formed as shown in reaction (1). The acetate ions underwent hydrolysis with water and released the hydroxide anions as shown in reaction (2). When ammonia solution was added, it formed ammonium and hydroxide ions, as shown in reaction (3) [18]. During the process, the zinc ions reacted with ammonium and hydroxide ions. Tetraamine zincate, zinc hydroxide, or tetrahydroxozincate were formed according to reactions (4–6). Due to dehydration of the complex in Foods 2019, 8, 88 5 of 10 the alkaline medium, ZnO nuclei are crystallized, as a result of the reaction of zinc hydroxide complex ions and tetraamine zincate complex ions, with hydroxide ions, as shown in reactions (8 and 9) [19].

3.2. Characterization of ZnO Microstars

3.2.1. FTIR Spectra Analysis The FTIR spectrum of the XG and ZnO microstars in the region of 450–4000 cm−1 is shown inFoods Figure 2019, 28,. x TheFOR majorPEER REVIEW absorption bands present in the FTIR spectrum of pure XG were shown5 of in 10 Foods 2019, 8, x FOR PEER REVIEW 5 of 10 Figure2a. The broad absorption peak at 3419 cm −1 was due to the stretching vibrations of O–H −1 band of 2920 cm could be assigned−1 to the absorption of symmetrical and asymmetrical stretching of groups. The band−1 of 2920 cm could be assigned to the absorption of symmetrical and asymmetrical band of 2920 cm could be assigned to the absorption of symmetrical and asymmetrical−1 stretching of methyl and methylene groups in XG. The peaks at 1731 and 1622 cm could−1 be due to the stretching of methyl and methylene groups in XG. The peaks at 1731 and 1622−1 cm could be due to methylcharacteristic and methyleneasymmetrical groups stretch in ofXG. the The carboxylate peaks at group 1731 andand the1622 carbonyl cm could group, be respectively. due to the the characteristic asymmetrical stretch of the carboxylate group and the carbonyl group, respectively. characteristic asymmetrical-1 stretch of the carboxylate group and the carbonyl group, respectively. The peak at 1419 cm− 1was assigned to the absorption of symmetrical stretching of the carboxyl group. The peak at 1419 cm -1 was assigned to the absorption of symmetrical stretching of the carboxyl group. The peak at 1419 cm was assigned to the absorption of symmetrical stretching of the carboxyl group.−1 Figure 2b shows the FTIR spectra of the XG-capped ZnO microstars. The band located at 555 cm−1 Figure2b shows the FTIR spectra of the XG-capped ZnO microstars. The band located at 555 cm −1 Figure 2b shows the FTIR spectra of the XG-capped ZnO microstars. The band located at 555 cm−1 correlated to the stretching mode of ZnO. In the presence of ZnO microstars the band at 3419 cm−1 correlated to the stretching mode of ZnO. In the presence of ZnO microstars the band at 3419 cm −1 correlated to the stretching−1 mode of ZnO. In the presence of ZnO microstars the band at 3419 cm shifted to 3378 cm−1, indicating the interaction of Zn with the OH group of XG. The interactions shifted to 3378 cm −1 , indicating the interaction of Zn with the OH group of XG. The interactions shiftedamong theto 3378resultant cm ZnO, indicating nanoparticles, the interaction and oxygen of Zn atoms with of thehydroxyl, OH group carboxyl, of XG. and The carbonyl interactions group among the resultant ZnO nanoparticles, and oxygen atoms of hydroxyl, carboxyl, and carbonyl group amongled to corresponding the resultant ZnO changes nanoparticles, in the positions, and oxygen and in atoms the strengths of hydroxyl, of spectrum carboxyl, of and the carbonyl XG. group ledled toto correspondingcorresponding changeschanges inin thethe positions,positions, andand inin thethe strengthsstrengths ofof spectrumspectrum ofof thethe XG.XG.

Figure 2. Fourier transform infrared spectroscopy (FTIR) spectra of (a) XG and (b) XG-capped ZnO Figure 2. Fourier transform infrared spectroscopy (FTIR) spectra of (a) XG and (b) XG-capped Figure 2. Fourier transform infrared spectroscopymicrostars. (FTIR) spectra of (a) XG and (b) XG-capped ZnO ZnO microstars. microstars. 3.2.2. XRD Analysis 3.2.2. XRD Analysis To conﬁrmconfirm thethe puritypurity and to determine the crystal structure of the particle particles,s, X-ray diffractometry To confirm the purity and to determine the crystal structure of the particles, X-ray diffractometry waswas used. As can be concluded from Figure3 3,, all all thethe diffractiondiffraction peaks peaks areare wellwell indexedindexed asas hexagonalhexagonal was used. As can be concluded from Figure 3, all the diffraction peaks are well indexed as hexagonal wurtzite-structureswurtzite-structures (JCPDS (JCPDS card card No. No. 36–1451), 36–1451), and and no nocharacteristic characteristic peaks peaks were were observed observed other other than wurtzite-structures (JCPDS card No. 36–1451), and no characteristic peaks were observed other than thanZnO. ZnO.The Thestrong strong and andnarrow narrow diffraction diffraction peaks peaks illustrated illustrated that that the the nanoparticles nanoparticles enjoyed enjoyed highhigh ZnO. The strong and narrow diffraction peaks illustrated that the nanoparticles enjoyed high crystallinity and purity.purity. crystallinity and purity.

Figure 3. X-ray diffraction (XRD) spectra of XG-capped ZnO microstars. Figure 3. X-ray diffraction (XRD) spectra of XG-capped ZnO microstars. 3.2.3. TEM Analysis 3.2.3. TEM Analysis The size, distribution, shape, and morphology of XG-capped ZnO microstars were evaluated by TEMThe (Figure size, 4a).distribution, The micrograph shape, and was morphology analyzed using of XG-capped the ImageJ ZnO software microstars version were 1.47v, evaluated an image by TEManalysis (Figure software 4a). Thedeveloped micrograph by the was NIH analyzed (http://imagejnihgov/ij/).The using the ImageJ software particle version size histogram,1.47v, an image with analysisnormal distribution software developed is provided by thein Figure NIH (http://imagejnihgov/ij/).The 4b. The prepared particles were particle starlike size microstructures,histogram, with normalranging distributionfrom 140–553 is nmprovided in diameter, in Figure with 4b. a meanThe prepared particle size particles of 358 were nm. starlike microstructures, ranging from 140–553 nm in diameter, with a mean particle size of 358 nm.

Foods 2019, 8, 88 6 of 10

3.2.3. TEM Analysis The size, distribution, shape, and morphology of XG-capped ZnO microstars were evaluated by TEM (Figure4a). The micrograph was analyzed using the ImageJ software version 1.47v, an image analysis software developed by the NIH (http://imagejnihgov/ij/).The particle size histogram, with normal distribution is provided in Figure4b. The prepared particles were starlike microstructures, rangingFoods 2019 from, 8, x FOR 140–553 PEER REVIEW nm in diameter, with a mean particle size of 358 nm. 6 of 10

FigureFigure 4. (a) Transmission electronelectron microscopymicroscopy (TEM)(TEM) micrographmicrograph ofof XG-cappedXG-capped ZnOZnO microstarsmicrostars andand ((bb)) correspondingcorresponding histogramhistogram withwith normalnormal distribution.distribution.

ShapeShape andand size size of of the the prepared prepared particles particles can becan dictated be dictated by the by presence the presence of XG inof the XG synthesis in the synthesis reaction. · Usingreaction. a similar Using method, a similar Hosseini-Sarvari method, Hosseini-Sarvari et al. [20] obtained et al ZnO. [20] nano-rods obtained from ZnO Zn(OAc) nano-rods2 2H2O from and NH4OH (25%), in the presence of the water-soluble linear polymer (PEG 2000), instead of XG. The presence Zn(OAc)2·2H2O and NH4OH (25%), in the presence of the water-soluble linear polymer (PEG 2000), ofinstead PEG 2000 of XG. was The found presence to affect of both PEG the 2000 shape was and foun the sized to of affect the resulting both the ZnO shape particles. and the It was size reported of the thatresulting the availability ZnO particles. of surfactant It was reported PEG 2000 that contributed the availability to the formationof surfactant of hexagonal PEG 2000 wurtize-structuredcontributed to the nanorodformation particles of hexagonal with a crystallitewurtize-structured size of 17–20 nanorod nm. particles with a crystallite size of 17‒20 nm.

3.3. Microbicidal Activity of Fabricated XG-Capped ZnO Microstars Although ZnO nanoparticles have great potential as a competitive zinc supplement, previous studies showed that the ZnO nanoparticles have an adverse effect on the growth and metabolism of different microorganisms. This antimicrobial characteristic significantly decreased the effectiveness, and functionality of ZnO nanoparticles, as they inhibited the gastrointestinal microbiota. Surface coating with biocompatible XG and increasing the particle size to micro scales may offer a better solution to address the negative effect of ZnO particles on the microorganisms. Therefore, the antibacterial activity of the XG-capped ZnO microparticles against both Gram-positive and Gram- negative bacteria were further investigated using the agar diffusion method. As shown in Figure 5, the presence of Ampicillin as a control resulted in an inhibition zone in all the strains. Among the investigated bacteria, B. lcheniformis showed the least inhibition zone (11 mm). The largest inhibition

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3.3. Microbicidal Activity of Fabricated XG-Capped ZnO Microstars Although ZnO nanoparticles have great potential as a competitive zinc supplement, previous studies showed that the ZnO nanoparticles have an adverse effect on the growth and metabolism of different microorganisms. This antimicrobial characteristic signiﬁcantly decreased the effectiveness, and functionality of ZnO nanoparticles, as they inhibited the gastrointestinal microbiota. Surface coating with biocompatible XG and increasing the particle size to micro scales may offer a better solution to address the negative effect of ZnO particles on the microorganisms. Therefore, the antibacterial activity of the XG-capped ZnO microparticles against both Gram-positive and Gram-negative bacteria were further investigated using the agar diffusion method. As shown in

FoodsFigure 20195,, 8 the, x FOR presence PEER REVIEW of Ampicillin as a control resulted in an inhibition zone in all the strains.7 of 10 Among the investigated bacteria, B. lcheniformis showed the least inhibition zone (11 mm). The largest zoneinhibition was observed zone was for observed B. subtilis for (32.5B. subtilis mm), (32.5followed mm), by followed B. sphaericus by B. (24 sphaericus mm) and(24 E. mm) coli (14.5 and E.mm). coli However,(14.5 mm). the However, agar plates the agarfor the plates different for the concentr differentations concentrations of XG-capped of XG-capped ZnO microstars ZnO microstars did not show did anynot showinhibition any inhibitionzones (Figure zones 5), (Figure which5), implied which impliedthat the that synthesized the synthesized particles particles had no had negative no negative effect oneffect the onbacterial the bacterial growth growth and metabolism. and metabolism.

FigureFigure 5. XG-cappedXG-capped ZnO ZnO microparticles microparticles and and Ampicillin Ampicillin against against ( (aa)) BacillusBacillus subtilis subtilis ((bb)) Bacillus sphaericussphaericus ((cc)) BacillusBacillus licheniformis and ( d)) Escherichia coli.

PreviousPrevious studies studies have have reported reported high high antibacteria antibacteriall activities activities for for naked naked ZnO ZnO nanoparticles nanoparticles against against bothboth Gram-positive Gram-positive and and Gram-negative Gram-negative bacteria bacteria [21]. [21]. For For instance, instance, it it was was reported reported that that the the growth inhibitioninhibition of of bacteria bacteria increased increased with with an an increase increase in in naked naked ZnO ZnO nanoparticle nanoparticle concentrations concentrations [22]. [22 It]. wasIt was noted noted that that the the size size of the of theinhibition inhibition zone zone was was dependent dependent upon upon the type the typeof bacteria, of bacteria, the size, the size,and theand concentrations the concentrations of ZnO of ZnO nanoparticles nanoparticles [23]. [23 Although]. Although the the exact exact mechanism mechanism of of ZnO ZnO antibacterial antibacterial activityactivity is stillstill notnot clear,clear, the the literature literature has has determined determined hypothetical hypothetical mechanisms mechanisms that that could could explain explain this thisphenomena. phenomena. One One of the ofhypothetical the hypothetical mechanisms mechanis isms the is formation the formation of reactive of reactive oxygen oxygen species species (ROS). (ROS).Studies Studies have shown have thatshown aquatic that ZnOaquatic nanoparticles ZnO nanoparticles produced produced an augmented an augmented level of ROS, level which of ROS, has whichbeen reported has been as reported the major as cause the major of nontoxicity. cause of nontoxicity. This increased This level increased was due level to the was generation due to the of generationhydrogen peroxideof hydrogen (H2 Operoxide2) molecules, (H2O2) which molecules, were which capable were of entering capable theof entering membrane, the membrane, where they wherecould damagethey could or kill damage the bacteria or kill [ 24the]. bacteria Another [2 proposed4]. Another mechanism proposed was mechanism the release was of zinc the ionsrelease (Zn 2+of) zinc ions (Zn2+) and their adhesion to the cell membrane, which caused mechanical damage to the cell wall. A further suggestion of the mechanism of ZnO antibacterial activity was the disruption of the membrane, resulting in membrane dysfunction and the internalization of ZnO nanoparticles into the bacteria [25]. In the present study we reported the successful fabrication of XG-capped ZnO microstars with no antibacterial activity. This behavior could be justified by an increase in the size of the prepared particles from the nano to the micro scale. It was obvious that the antimicrobial properties of the metallic particles were severely influenced by the particle size and shape [14,15]. Increases in the particle size usually resulted in a decrease of the antimicrobial properties. However, the antimicrobial properties of metallic particles were not only under the control of particle size, and particle size was not the dominant parameter. Attention should be paid to the other physical and chemical properties of the particles [15]. Surface capping, and using a biocompatible material, could minimize the inhibitory effects of metallic particles on bacterial growth. XG is a particular exopolysaccharide secreted by the fermentation of the bacterium Xanthomonas campestris in aerobic conditions, from

Foods 2019, 8, 88 8 of 10 and their adhesion to the cell membrane, which caused mechanical damage to the cell wall. A further suggestion of the mechanism of ZnO antibacterial activity was the disruption of the membrane, resulting in membrane dysfunction and the internalization of ZnO nanoparticles into the bacteria [25]. In the present study we reported the successful fabrication of XG-capped ZnO microstars with no antibacterial activity. This behavior could be justified by an increase in the size of the prepared particles from the nano to the micro scale. It was obvious that the antimicrobial properties of the metallic particles were severely influenced by the particle size and shape [14,15]. Increases in the particle size usually resulted in a decrease of the antimicrobial properties. However, the antimicrobial properties of metallic particles were not only under the control of particle size, and particle size was not the dominant parameter. Attention should be paid to the other physical and chemical properties of the particles [15]. Surface capping, and using a biocompatible material, could minimize the inhibitory effects of metallic particles on bacterial growth. XG is a particular exopolysaccharide secreted by the fermentation of the bacterium Xanthomonas campestris in aerobic conditions, from sugar cane, corn or their derivatives [26]. It is a natural heteropolysaccharide, with branched chains and acidic characteristics, consisting of repeated pentasaccharide units with two glucose units, two mannose units, and one glucuronic acid unit in the ratio of 2.8:2.0:2.0 [27]. There are two proposed mechanisms for the stabilization of inorganic particles using natural gums. The first mechanism was the result of XG adsorbed through the surface of particles, which caused steric repulsion among the particles. The second mechanism involved XG increasing the viscosity of the particle suspensions, hence gradually decreasing the aggregation processes [28,29]. XG was highly soluble in both hot and cold water, hydrated quickly, and was stable over a wide range of temperature, acidic, and alkaline conditions [30,31]. XG has a wide range of applications in the food industry due to its nontoxic and biocompatible properties, thickening properties, and high suspending ability [31–34]. Therefore, the XG-capped ZnO microstars that were synthesized in this study may be a possible approach in creating a dietary zinc supplement that could facilitate zinc intake in the future.

4. Conclusions A green and -efﬁcient method for the synthesis of XG-capped ZnO microstars was developed using XG as the stabilizing agent. TEM, XRD, and FTIR were used to characterize the fabricated nanoparticles. The antibacterial activity of the fabricated XG-capped ZnO microstars were also investigated using the agar diffusion method. It was observed that all tested strains were susceptible to Ampicillin as a control. However, different concentrations of XG-capped ZnO microstars did not show any inhibitory effects against both Gram-positive and Gram-negative bacteria. This behavior could be justiﬁed by the particles at the micro scale, and the presence of biocompatible XG capping on ZnO microstars.

Author Contributions: A.E., S.-M.T., M.S., and A.B. contributed to the conceptualization. A.E., F.M., S.-M.T., M.S., C.B., and A.B. contributed to the methodology. A.E., S.-M.T., M.S., C.B., and D.N. conducted the investigation, validation, and formal analysis. A.E., F.M., M.S., and C.B. contributed to the original draft preparation. A.E., Y.G., and A.B. contributed to the review and editing of the manuscript. Y.G. and A.B. contributed to funding acquisition. Funding: This study was financially supported under a collaborative agreement by the Shiraz University of Medical Sciences, Shiraz, Iran and the University of Waikato, New Zealand. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Broadley, M.R.; White, P.J. Eats roots and leaves. Can edible horticultural crops address dietary calcium, magnesium and potassium deﬁciencies? Proc. Nutr. Soc. 2010, 69, 601–612. [CrossRef][PubMed] 2. Hambidge, M. Zinc and Health: Current Status and Future Directions. J. Nutr. 2000, 130, 1341s–1519s. [CrossRef][PubMed] 3. World Health Organization. Vitamin and Mineral Requirements in Human Nutrition; World Health Organization: Geneva, Switzerland, 2004. Foods 2019, 8, 88 9 of 10

4. Nutritious Food for All in a Changing World. Cell 2014, 157, 1493–1495. [CrossRef] 5. Gregory, P.J.; Wahbi, A.; Adu-Gyamfi, J.; Heiling, M.; Gruber, R.; Joy, E.J.M.; Broadley, M.R. Approaches to reduce zinc and iron deficits in food systems. Glob. Food Secur. 2017, 15, 1–10. [CrossRef] 6. Hassan, M. Zinc Oxide Nanoparticles Fortified Biscuits as a Nutritional Supplement for Zinc Deficient Rats. J. Nanomed. Res. 2016, 4, 81. [CrossRef] 7. Roohani, N.; Hurrell, R.; Kelishadi, R.; Schulin, R. Zinc and its importance for human health: An integrative review. J. Res. Med. Sci. 2013, 18, 144–157. [PubMed] 8. IZiNCG. International Zinc Nutrition Consultative Group (IZiNCG) technical document# 1. Assessment of the risk of zinc deficiency in populations and options for its control. Food Nutr. Bull. 2004, 25, S99–S203. 9. Chaumeil, J. Micronization: A method of improving the bioavailability of poorly soluble drugs. Methods Find. Exp. Clin. Pharmacol. 1998, 20, 211–216. [PubMed] 10. Ramesh, M.; Anbuvannan, M.; Viruthagiri, G. Green synthesis of ZnO nanoparticles using Solanum nigrum leaf extract and their antibacterial activity. Spectrochim. Acta Mol. Biomol. Spectrosc. 2015, 136, 864–870. [CrossRef][PubMed] 11. Banoee, M.; Seif, S.; Nazari, Z.E.; Jafari-Fesharaki, P.; Shahverdi, H.R.; Moballegh, A.; Moghaddam, K.M.; Shahverdi, A.R. ZnO nanoparticles enhanced antibacterial activity of ciprofloxacin against Staphylococcus aureus and Escherichia coli. J. Biomed. Mater. Res. B Appl. Biomater. 2010, 93, 557–561. [CrossRef][PubMed] 12. Ambika, S.; Sundrarajan, M. Antibacterial behaviour of Vitex negundo extract assisted ZnO nanoparticles against pathogenic bacteria. J. Photochem. Photobiol. B 2015, 146, 52–57. [CrossRef][PubMed] 13. Thursby, E.; Juge, N. Introduction to the human gut microbiota. Biochem. J. 2017, 474, 1823–1836. [CrossRef] [PubMed] 14. Pal, S.; Tak, Y.K.; Song, J.M. Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the gram-negative bacterium Escherichia coli. Appl. Environ. Microbiol. 2007, 73, 1712–1720. [CrossRef][PubMed] 15. Wang, L.; Hu, C.; Shao, L. The antimicrobial activity of nanoparticles: Present situation and prospects for the future. Int. J. Nanomed. 2017, 12, 1227. [CrossRef][PubMed] 16. Balouiri, M.; Sadiki, M.; Ibnsouda, S.K. Methods for in vitro evaluating antimicrobial activity: A review. J. Pharm. Anal. 2016, 6, 71–79. [CrossRef][PubMed] 17. Kołodziejczak-Radzimska, A.; Jesionowski, T. Zinc Oxide—From Synthesis to Application: A Review. Materials 2014, 7, 2833–2881. [CrossRef][PubMed] 18. Pung, S.-Y.; Lee, W.-P.; Aziz, A. Kinetic study of organic dye degradation using ZnO particles with different morphologies as a photocatalyst. Int. J. Inorg. Chem. 2012, 2012, 608183. [CrossRef] 19. Wallace, I.; Eshu, O.V.; Chukwunonso, O.B.; Okoro, U.C. Synthesis and characterization of zinc oxide (ZnO) nanowire. J. Nanomed. Nanotechnol. 2015, 6, 2. 20. Hosseini-Sarvari, M.; Tavakolian, M. Preparation, characterization, and catalysis application of nano-rods zinc oxide in the synthesis of 3-indolyl-3-hydroxy oxindoles in water. Appl. Catal. A Gen. 2012, 441, 65–71. [CrossRef] 21. Xie, Y.; He, Y.; Irwin, P.L.; Jin, T.; Shi, X. Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni. Appl. Environ. Microbiol. 2011, 77, 2325–2331. [CrossRef] [PubMed] 22. Gunalan, S.; Sivaraj, R.; Rajendran, V. Green synthesized ZnO nanoparticles against bacterial and fungal pathogens. Prog. Nat. Sci. Mater. Int. 2012, 22, 693–700. [CrossRef] 23. Emami-Karvani, Z.; Chehrazi, P. Antibacterial activity of ZnO nanoparticle on Gram-positive and Gram-negative bacteria. Afr. J. Microbiol. Res. 2011, 5, 1368–1373. 24. Sawai, J.; Kawada, E.; Kanou, F.; Igarashi, H.; Hashimoto, A.; Kokugan, T.; Shimizu, M. Detection of Active Oxygen Generated from Ceramic Powders Having Antibacterial Activity. J. Chem. Eng. Jpn. 1996, 29, 627–633. [CrossRef] 25. Sirelkhatim, A.; Mahmud, S.; Seeni, A.; Kaus, N.H.M.; Ann, L.C.; Bakhori, S.K.M.; Hasan, H.; Mohamad, D. Review on Zinc Oxide Nanoparticles: Antibacterial Activity and Toxicity Mechanism. Nano-Micro Lett. 2015, 7. [CrossRef][PubMed] 26. Barrere, G.; Barber, C.; Daniels, M. Molecular cloning of genes involved in the production of the extracellular polysaccharide xanthan by Xanthomonas campestris pv. campestris. Int. J. Biol. Macromol. 1986, 8, 372–374. [CrossRef] Foods 2019, 8, 88 10 of 10

27. Becker, A.; Katzen, F.; Pühler, A.; Ielpi, L. Xanthan gum biosynthesis and application: A biochemical/genetic perspective. Appl. Microbiol. Biotechnol. 1998, 50, 145–152. [CrossRef][PubMed] 28. Comba, S.; Dalmazzo, D.; Santagata, E.; Sethi, R. Rheological characterization of xanthan suspensions of nanoscale iron for injection in porous media. J. Hazard. Mater. 2011, 185, 598–605. [CrossRef][PubMed] 29. Xue, D.; Sethi, R. Viscoelastic gels of guar and xanthan gum mixtures provide long-term stabilization of iron micro-and nanoparticles. J. Nanopart. Res. 2012, 14, 1239. [CrossRef] 30. Sereno, N.M.; Hill, S.E.; Mitchell, J.R. Impact of the extrusion process on xanthan gum behaviour. Carbohydr. Res. 2007, 342, 1333–1342. [CrossRef][PubMed] 31. Sharma, B.; Naresh, L.; Dhuldhoya, N.; Merchant, S.; Merchant, U. Xanthan gum—A boon to food industry. Food Promot. Chron. 2006, 1, 27–30. 32. Rosalam, S.; England, R. Review of xanthan gum production from unmodiﬁed starches by Xanthomonas comprestris sp. Enzym. Microb. Technol. 2006, 39, 197–207. [CrossRef] 33. Benny, I.S.; Gunasekar, V.; Ponnusami, V. Review on application of xanthan gum in drug delivery. Int. J. PharmTech Res. 2014, 6, 1322–1326. 34. Palaniraj, A.; Jayaraman, V. Production, recovery and applications of xanthan gum by Xanthomonas campestris. J. Food Eng. 2011, 106, 1–12. [CrossRef]

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